Screening for components involved in NLR-mediated immune signalling

Abstract

Many Members of the intracellular nucleotide binding and oligomerisation domain(NOD)-like receptor (NLR) family have functions in the innate immune system. The NLR NOD1 acts as pattern recognition receptor and confers immune responses against a broad range of bacteria by triggering NF-kappaB and MAPK signalling cascades upon detection of bacterial peptidoglycan. This contributes to bacterial clearance, to the onset of a pro-inflammatory immune response and the release of anti-microbial peptides. As overwhelming inflammatory responses can be detrimental to the host, inflammatory signalling cascades have to be tightly controlled. Even though the main components of the NOD1 signalling cascade are identified,little is known about the regulation and fine-tuning so far. In this project, we identified novel components of the NOD1 signalling pathway by an auto-mated high-throughput siRNA screen using a cell based NF-kappaB reporter system in epithelial HEK293T cells. To this end,we screened the human druggable genome siRNA library for NOD1-mediated NF-kappaB activation upon stimulation with the elicitor Tri-DAP. Hits specifically involved in NOD1-mediated NF-kappaB activation were identified using TNF-α-stimulation as differential read-out. Finally, these hits were validated in myeloid THP1 cells. Beside the established NOD1 pathway component RIP2, the combined screening steps identified the BIRC family member XIAP as the strongest inhibiting hit. Follow-up experiments confirmed XIAP as an essential component of NOD1-mediated responses to the minimal NOD1 elicitor and to Shigella flexneri. We also revealed that XIAP contributes to responses mediated by the closely related NOD2 protein. In line with a recent report, we provide evidence that XIAP acts upstream of the IKK complex in the NOD1 signalling cascade. Strikingly, the screen revealed that the type II BMP receptor BMPR-2 is specifically involved in NOD1 signalling. Further experiments confirmed these findings and revealed that BMPR-2 positively regulates NOD1 signalling, likely by contributing to stabilisation of XIAP. Furthermore, we analysed the contributions of the BIRC proteins BIRC2 and BIRC3, which are closely related to XIAP, and of the other human BIRC proteins to NOD1-mediated responses. By using siRNA-mediated gene knock-down, we confirmed that BIRC2 positively regulates NOD1 signalling. Furthermore, we provide evidence that also BIRC5 and BIRC8, which have not been linked to innate immunity so far, positively contribute to NOD1-mediated inflammatory responses. Another event in NOD signalling that is still not fully understood is how bacterial peptidoglycan is translocated to the cytoplasm of host cells, as extracellular bacteria are known to activate NOD1 as well as NOD2. In the second part of this project, we analysed if bacterial outer-membrane vesicles (OMVs) might serve as carriers for peptidoglycan. We provide clear evidence that OMVs derived from the extracellular pathogen Vibrio cholerae are internalised by host cells, contain peptidoglycan and trigger NOD1- and NOD2-dependent inflammatory responses. OMVs derived from bacteria deficient for HapR, a master regulator of quorum sensing that represses the expression of virulence genes, induced markedly lower NOD-mediated inflammatory responses than OMVs derived from WT bacteria. In contrast, the overall peptidoglycan composition and the inflammatory potential of bacterial lysates derived from deltahapR compared to WT bacteria did not change. We conclude that V. cholerae uses quorum sensing to influence the pepdidoglycan content of OMVs, to prevent detection by the host innate immune system under virulent conditions.